CC Lec Mod 3 Unit 4

• They indicate the value that is expected in 95% of healthy individuals during routine medical tests.
• Chromatography refers to the group of techniques used to separate complex mixtures based on physical and chemical interactions between compounds and the stationary phase
• In chromatography, the mobile phase carries the sample through a bed, layer, or column containing the stationary phase
• Chromatography has four basic components: the mobile phase, the stationary phase, the column, and the eluate
• The bases of separation in chromatography are the different physical and chemical interactions between compounds and the stationary phase
• There are two basic forms of chromatography: Planar chromatography and column chromatography
• Planar chromatography includes paper chromatography and thin-layer chromatography (TLC)
• Column chromatography includes gas chromatography (GC) and liquid chromatography (LC)
• Chromatographic separations are classified into ion-exchange, partition, adsorption, size-exclusion, and affinity mechanisms
• Most clinical applications use chromatographic separations based on ion-exchange and partition mechanisms
• Separation of solutes is based on differences in their ionic charge or the magnitude of their ionic charges
• The stationary phase in this technique is the particle surfaces of a plastic resin or silica coated or bound with functional groups with fixed cationic or anionic charges
• An exchangeable ion, called the counterion, is found in close proximity to the fixed charge and solute ions in the mobile phase exchange with the counterions to maintain electrochemical neutrality
• Solute ions are eluted selectively by changing the mobile phase pH, ionic strength, or both
• Cation-exchange particles contain negatively charged functional groups and are used to separate or "exchange" cationic solutes, while anion-exchange packings are used to separate anionic solutes
• Clinical applications of this technique include the separation of amino acids, peptides, proteins, nucleotides, oligonucleotides, and nucleic acids
• Separation of nucleic acids and proteins primarily depends on the sign and ionic charge density
• Partition Chromatography involves the separation of solutes based on their differential distribution between two immiscible liquids
• One of the immiscible liquids serves as the stationary phase, prepared by adsorbing or chemically bonding a thin film of the liquid onto the surface of support particles or onto the inner wall of a capillary column
• Separation is based on differences in the relative solubility of solute molecules between the stationary and mobile phases
• Partition chromatography is classified as gas-liquid chromatography (GLC) or liquid-liquid chromatography (LLC), with LLC further categorized as normal phase or reversed phase
• Hydrophilic interaction chromatography (HILIC) is a version of partition chromatography used for separating biological, organic, and inorganic molecules by differences in polarity
• In Adsorption Chromatography, separation is based on the differences between the adsorption and desorption of solutes at the surface of a solid
• The sample and the mobile phase compete for the adsorptive sites on the solid stationary phase such as silica or alumina
• Molecules that are most soluble in the mobile phase move fastest, while the least soluble move slowest
• Size-exclusion Chromatography separates solutes based on their molecular sizes and shape
• A variety of materials are used as stationary phases, including cross-linked dextran, polyacrylamide, agarose, polystyrene-divinylbenzene, porous glass, and combinations of those
• Hydrophilic gel like dextran and agarose is used in the separation of enzymes, antibodies, and proteins in a process called Gel Filtration
• Affinity Chromatography uses the unique and specific biological interaction of the analyte and ligand for separation
• Enzyme-substrate, hormone-receptor, or antigen-antibody interactions are used in this type of chromatography
• Affinity chromatography has been used in the clinical laboratory to separate analytes such as glycated hemoglobin and low-density lipoproteins
• Chromatographic Procedures:

  • Two forms of chromatography: Planar and Column chromatography
  • Planar chromatography includes Paper Chromatography and Thin-Layer Chromatography
• In Paper Chromatography, the stationary phase consists of a layer of water or a polar solvent coated onto the fibers of a sheet of paper
• Paper chromatography uses a stationary phase of water or a polar solvent coated onto paper fibers, typically Whatman paper
• Separation in paper chromatography is normal-phase partition chromatography between the nonpolar mobile phase and the polar stationary phase
• Paper chromatography is used for the fractionation of sugar and amino acids
• Affinity chromatography uses the "lock-and-key binding" principle for separation
• Thin-layer chromatography (TLC) involves applying the sample as a small spot or band near the bottom edge of a plate
• In TLC, solutes can be visualized directly if they are colored or fluorescent, or by performing autoradiography for radioactive compounds
• Migration of solutes in paper chromatography (PC) and TLC is expressed as a Retention factor (Rf) value